Milk is one of the most thoroughly researched foods in history. Countless scientific papers document milk's composition and describe the biological functionalities in this complex bio-resource. Proteins, peptides, enzymes and other biomolecular substances constitute a major and very important fraction in milk and are believed responsible for many of the specific functionalities passed on from a mother to her new-born in addition to basic nutrients.
During the past two decades, there has been significant focus on utilisation of bovine whey proteins. Today, several bovine Whey Protein Concentrates (WPC) and bovine Whey Protein Isolates (WPI) are standard products obtained through various membrane filtration techniques as well as ion exchange adsorption procedures. Further utilisation of the bovine whey in terms of fractionation of the proteins into individual protein fractions, such as β-lactoglobulin, α-lactalbumin, immunoglobulins, lactoperoxidase, and lactoferrin, is made possible through chromatographic packed bed separation techniques. Protein products from chromatographic separation technologies are generally characterised by their low- to non-fat content and are useful for a broad range of applications e.g. within food, feed, functional foods, and health care products.
Since the first market introductions of WPC and WPI products and more recently the first purified single protein products (e.g. lactoferrin and lactoperoxidase) there has evolved an ever increasing demand for even more sophisticated and still more efficient and cost effective productions methods.
Among the various industrial chromatographic separation techniques developed in recent years, Expanded Bed Adsorption (EBA) has been successfully introduced to the certain fields of biotechnology industry. EBA is a type of fluidised bed adsorption wherein the level of back-mixing is kept at a minimum. Compared with other chromatographic separation technologies, EBA offer a significant advantage because it can be used directly with non-clarified feed.
During EBA, the adsorbent bed is allowed to expand inside the column when a flow of liquid is applied (see FIG. 1). Expansion/fluidisation of the bed is often effected in a column having provided at each of its ends a net structure covering the cross-sectional area of the column, or some other perforated devices, which will not generate turbulence in the flow. See, for instance, WO-A-9218237 (Amersham Pharmacia Biotech AB, Sweden). The same effect has also been observed in a system utilising a stirred inlet flow WO-A-9200799, (UpFront Chromatography A/S). In addition, other distributors are likely to be feasible.
In the expanded bed state, the distance between the adsorbent particles result in a free passage of particulate impurities in the feed stream. By contrast, traditional packed beds work as depth filters that can clog, resulting in increased back-pressure unless the feed is thoroughly clarified. Since no significant pressure builds up in an EBA column, it is possible to apply EBA without the limitations in size and flow rate normally associated with packed-bed columns.
An EBA process is characterised by very limited back-mixing of the liquid inside the column as opposed to the well know turbulent fluidised beds typically employed for chemical reactions. Back-mixing in a bed is often measured as axial dispersion (“vessel dispersion number”), see Levenspiel, “Chemical Reaction Engineering” 2nd Edition, John Wiley & Sons (1972).
The adsorbent media employed in an EBA process must have a higher density than the feed stock in order to produce acceptable flow rates during operation. If the density is too low, the media will be lost in the column effluent. Generally, EBA adsorbent particles may either be designed to be impermeable to the fluid, in which case the available surface area per unit volume is small; or particles may be designed to be permeable to the fluid, in which case the material chosen has to have the correct density per se. Unfortunately, the most interesting materials for many applications, e.g. materials such as natural and synthetic polysaccharides like agar, alginates, carrageenans, agarose, dextran, modified starches, and celluloses; synthetic organic polymers and copolymers typically based on acrylic monomers used for chromatographic purification of proteins in packed bed columns are not of suitable density per se. Therefore, these materials are not readily applied in EBA.
However, certain types of organic polymers and certain types of silica based materials may be produced to provide carrier particles of suitable density, but such carriers may not at the same time be suitable adsorbents, e.g. for protein purification procedures, where such materials may provide low permeability, non-specific interactions and denature bound proteins. Further, for such polymers, it may be difficult and expensive to design derivatisation schemes for affinity chromatography media. In addition, certain types of permeable silica particles have been used for EBA. However, the properties of these materials are far from optimal. Thus, the materials are unstable at pH above 7, fragile to shear forces, and provide non-specific interactions. In addition solid silicate materials have a maximal density of approx. 2.5 g/mL.
The density of the adsorbent media may be controlled by an inert, high-density core incorporated in the polymer phase (composite media, conglomerates see e.g. WO-A-9200799). High-density core materials are typically chosen from high density materials such as glass, quartz or heavy metals either in the form of an alloy such as stainless steel or an oxide (e.g. zirconium oxide) or some other metal salt (e.g. tungsten carbide). The core material may also comprise metal spheres (e.g. tantalum). The core material of the particles may vary in size and shape. Typical sizes are within 5-80 micrometers.
In EBA, as a result of the optimisation of the characteristics of the adsorbent media (size distribution), plug-flow conditions with very little back-mixing is obtained inside the column. The plug-flow behaviour is crucial in order to obtain an efficient adsorption.
Today, several important bio-pharmaceuticals are being produced using the EBA technology. However, no commercial processes for milk and whey fractionation are based on EBA so far. This is in great part due to the large scale of the process required for milk and whey fractionation, typically involving extremely high volumes of raw material to be treated per day (e.g. several m3/hour) which requires extremely high efficiency and productivity of the EBA system. Current processes are not capable, in practice, to achieve the level of performance for these and certain other raw materials.
A major supplier of EBA adsorbents and EBA columns is UpFront Chromatography A/S, Denmark. These products are supplied under the trademark FastLine (see e.g.WO 92/00799, UpFront Chromatography A/S, Denmark), which discloses a large number of fillers and polymeric materials that can be combined to produce composite beads, conglomerates) intended for adsorption in EBA.
Amersham Pharmacia Biotech AB, Sweden markets StreamLine which utilise porous beads of agarose with quartz particles as filler material (WO-A-9218237, Pharmacia Biotech AB).
Another supplier is Bioprocessing Ltd. (Durham, England) whose porous glass beads (Prosep0) can be used for chromatography on expanded beds (Beyzavi et al, Genetic Engineering News, Mar. 1, 1994 17).
WO 97/17132 (Amersham Pharmacia Biotech) discloses a population of beads having a density >1 g/cm3 and comprising a polymer base matrix in which a particulate filler is incorporated. The beads are characterized in that the filler particles have a density >3 g/cm3 and in that the density and/or size of the beads are distributed within predetermined density and size ranges. Particularly important filler particles are those which have rounded shapes, for instance spheres, ellipsoids or aggregates/agglomerates thereof. The bead population is particularly usable in adsorption processes in fluidized beds, with preference to stable expanded beds.
WO 00/57982 discloses a particulate material having a density of at least 2.5 g/mL, where the particles of the particulate material have an average diameter of 5-75 μm, and the particles of the particulate material are essentially constructed of a polymeric base matrix, e.g. a polysaccharide such as agarose, and a non-porous core material, e.g. steel and titanium, said core material having a density of at least 3.0 g/mL, said polymeric base matrix including pendant groups which are positively charged at pH 4.0 or which are affinity ligands for a bio-molecule. Possible pendant groups include polyethyleneimine (PEI), diethylaminoethyl (DEAE) and quaternary aminoethyl (QAE). The materials are useful in expanded bed or fluidised bed chromatography processes, in particular for purification of bio-macromolecules such as plasmid DNA, chromosomal DNA, RNA, viral DNA, bacteria and viruses.
WO-A-8603136 (Graves and Burns; University Patents Inc) discloses beads containing magnetic filler particles and their use in fluidized beds stabilized by an externally applied magnetic field. See also Burns et al., Biotechnol. Bioengin. 27 (1985) 137-145.
WO-A1-9833572 (Amersham Pharmacia Biotech) discloses a method for adsorption of a substance from a liquid sample on a fluidized bead or stirred suspension, in which the beads used comprise a base matrix and exhibit a structure having affinity to the substance, characterized in that the structure is covalently bound to the base matrix via an extender. Populations of beads in which the beads contain a filler incorporated in a base matrix and an extender are also described.
In chromatography on packed beds it has earlier been suggested to use porous beads, the pores of which wholly or partly have been filled with hydrophilic gels carrying affinity ligands, such as ion exchange groups. One example is Macrosob-K which is macroporous kieselguhr which has been filled with agarose which in turn has been derivatized to exhibit DEAE or CM ion exchange groups (Macrosorb-KAX.DEAE and Macrosorb KAX.CM, respectively (GB-A-1,586,364, Miles). This latter type of materials have also been applied in fluidized bed chromatography (Bite et al., In: Verrall et al., Separations for Biotechnology (1987), Ellis Horwood LTD, Chapter 13, 193-199).
U.S. Pat. No. 4,976,865 (Sanchez, et al, CNRS) teaches fluidised beds and the use of segmented columns to mimic the multi-step adsorption taking place in packed as well as stabilised expanded beds for isolation of whey compounds. The beads used in the experimental part are silica particles (Spherosil, density=1.4 g/mL, mean particle size=225 μm) that have been coated. The linear flow rate implemented in the experimental part is 1.3×10−3 m/s, which is equal to 468 cm/hour). The experimental parts discloses the use of this type of fluidized bed adsorption for separation of biological macromolecules from whey. There is no disclosure of any flow rates and/or binding capacities obtainable with adsorbents having a lower than 225 μm mean particle size.
Immunoglobulins—or antibodies—constitute a very important class of proteins which are present in various body fluids of mammals, birds and fish functioning as protective agents of the animal against substances, bacteria and virus challenging the animal. Immunoglobulins are typically present in animal blood, milk, and saliva as well as other body fluids and secretions.
All the above mentioned applications of immunoglobulins requires some sort of isolation of the antibody from the crude raw material, but each kind of application has its own very varying demands with respect to the final purity and allowable cost of the antibody product.
Generally, there exists a very broad range of different methods available for isolation of immunoglobulins giving a very broad range of final purities, yields and cost of the product.
Traditional methods for isolation of immunoglobulins are based on selective reversible precipitation of the protein fraction comprising the immunoglobulins while leaving other groups of proteins in solution. Typical precipitation agents being ethanol, polyethylene glycol, lyotropic (anti-chaotropic) salts such as ammonium sulfate and potassium phosphate, and caprylic acid.
Typically, these precipitation methods are giving very impure products while at the same time being time consuming and laborious. Furthermore, the addition of the precipitating agent to the raw material makes it difficult to use the supernatant for other purposes and creates a disposal problem. This is particularly relevant in relation to the large scale purification of immunoglobulins from for instance, whey.
Ion exchange chromatography is another well known method of protein fractionation frequently used for isolation of immunoglobulins. However, this method is not generally applicable because of the restraints in ionic strength and pH necessary to ensure efficient binding of the antibody together with the varying isoelectric points of different immunoglobulins.
Protein A and Protein G affinity chromatography are very popular and widespread methods for isolation and purification of immunoglobulins, particularly for isolation of monoclonal antibodies, mainly due to the ease of use and the high purity obtained. Although being popular it is however recognised that Protein A and Protein G poses several problems to the user among which are: very high cost, variable binding efficiency of different monoclonal antibodies (particularly mouse IgG1), leakage of Protein A/Protein G into the product, and low stability of the matrix in typical cleaning solutions, e.g. 1 M sodium hydroxide. Each of these drawbacks have its specific consequence in the individual application, ranging from insignificant to very serious and prohibitive consequences.
Hydrophobic chromatography is also a method widely described for isolation of immunoglobulins, e.g. in “Application Note 210, BioProcess Media” published by Pharmacia LKB Biotechnology, 1991. In this publication, a state of the art product “Phenyl Sepharose High Performance” is described for the purpose of purifying monoclonal antibodies from cell culture supernatants. As with other hydrophobic matrices employed so far it is necessary to add lyotropic salts to the raw material to make the immunoglobulin bind efficiently. The bound antibody is released from the matrix by lowering the concentration of lyotropic salt in a continuous or stepwise gradient. It is recommended to combine the hydrophobic chromatography with a further step if highly pure product is the object.
The disadvantage of this procedure is the necessity to add lyotropic salt to the raw material as this gives a disposal problem and thereby increased cost to the large scale user. The addition of lyotropic salts to the raw materials would in many instances be prohibitive in large scale applications as the salt would prevent any economically feasible use of the immunoglobulin depleted raw material in combination with the problem of disposing several thousand liters of waste.
Thiophilic adsorption chromatography was introduced by J. Porath in 1985 (J. Porath et al; FEBS Letters, vol.185, p.306, 1985) as a new chromatographic adsorption principle for isolation of immunoglobulins. Porath describes the technology wherein divinyl sulfone-activated agarose in combination with various ligands comprising a free mercapto-group demonstrate specific binding of immunoglobulins in the presence of 0.5 M potassium sulfate, i.e. a lyotropic salt. It was postulated that the sulfone group, from the vinyl sulfone spacer, and the resulting thio-ether in the ligand was a structural necessity to obtain the described specificity and capacity for binding of antibodies. It was, however, later shown that the thio-ether could be replaced by nitrogen or oxygen if the ligand further comprised an aromatic radical (K. L. Knudsen et al, Analytical Biochemistry, vol 201, p.170, 1992).
Although the matrices described for thiophilic chromatography generally show good performance, they also have a major disadvantage in that it is needed to add lyotropic salts to the raw material to ensure efficient binding of the immunoglobulin, which is a problem for the reasons discussed above.
Other thiophilic ligands coupled to epoxy activated agarose have been disclosed in (I. Porath et.al., Makromol. Chem., Makromol. Symp., vol. 17, p.359, 1988) and (A. Schwarz et.al., Journal of Chromatography B, vol. 664, pp. 83-88, 1995), e.g. 2-mercaptopyridine, 2-mercaptopyrimidine, and 2-mercaptothiazoline. However, all these affinity matrices still have inadequate affinity constants to ensure an efficient binding of the antibody without added lyotropic salts.
To avoid the above mentioned problems and disadvantages the investigators of the present invention have developed a method for large scale fractionation, purification and isolation of at least one protein from a protein-containing mixture. This method is applicable for industrial use, it can handle very large volumes of a protein-containing mixture, it is fast and it provides a highly purified protein.
The investigators of the present invention also found that it was possible to carry out the fractionation, purification and isolation of proteins without the use of lyotropic salts.